WO2006011580A1 - Method of purifying virus envelope - Google Patents

Abstract

It is intended to provide a method of industrially purifying the envelope of a virus (for example, Sendai virus (Hemagglutinating Virus of Japan, HVJ)). More specifically, it is intended to provide a method whereby an inactivated virus envelope is purified by the combined use of ion exchange chromatography with hydrophobic chromatography to thereby purify the envelope at a high yield while sustaining the cell fusion activity of the virus. The virus envelope thus purified is usable as a vector for transferring a biopolymer such as a gene into cells or living body. This method is also applicable to the purification of an attenuated envelope virus.

Description

 Specification

 Purification method of virus envelope

Technical field

 TECHNICAL FIELD The present invention relates to an industrial purification method of an envelope (for example, Hemagglutinating Virus of Japan, hereinafter referred to as HVJ). The purified virus envelope can be used as a vector for introducing a biopolymer such as a gene into a cell or a living body. Background art

 Many viral and non-viral methods have been developed for the purpose of introducing genes into cultured cells and biological tissues for gene function analysis and gene therapy (Mulligan, Science, 260, 926-932, 1993 and Ledley, Human Gene Therapy, Vol. 6, 1129-1144, 1995). In general, viral methods are effective for introducing genes into cells. However, the method using a viral vector is problematic in terms of safety because of the possibility of gene transfer and expression of the parent virus, its immunogenicity, and the modification of the host genome structure. On the other hand, many non-viral methods using ribosomes have lower cytotoxicity and immunogenicity than viral methods, but their gene transfer efficiency into cultured cells and biological tissues tends to be inferior to that of viral vectors.

HVJ is a virus belonging to the Paramyxoviridae family that has an envelope and hemaggluton and neuraminidase on its surface. HVJ is attracting attention as a fusion of Erich tumor cells (Okada, Ikeno Journal, 1, 10 3— 1 1 0, 1 9 5 8), and analysis of cell membrane fusion activity (hereinafter referred to as fusion activity) The use as a gene transfer vector has been studied. HVJ is highly immunogenic, and is known to induce CTL when NP protein is produced in large quantities (Cole GA Journal of Immunology 158, 4301-4309 (1997), and there is also a concern that the protein synthesis of the host may be inhibited. Therefore, a method for producing fusion particles (HVJ-ribosomes) by fusing liposomes encapsulating genes and proteins with HVJ that has been inactivated by UV irradiation in advance has been devised. Invasive gene transfer became possible (US Pat. No. 5,631,237, Dzau et al., Proc. Natl. Acad. Sci. USA, 93, 1 1421-11425, 1996 and Kaneda et al., Mo. l ecul ar Medicine Today, 5, 298-303, 1999). However, this method has the complication of having to prepare two different vesicles, quinoles and ribosome, and HVJ-liposomes have an average diameter 1.3 times larger than HVJ particles, which is more fusion activity than HVJ alone. Was found to be reduced to one-tenth or less.

 Kaneda has developed a highly safe HVJ vector that introduces genes into cells with high efficiency (W001 / 57204). That is, it inactivates the genome of HVJ and other envelopeloinoses, encapsulates a biopolymer such as a gene in its envelope, and uses it as a vector into cells or in vivo. However, at this stage, an efficient purification method for the HVJ envelope has not been established.

 Kaneda has also developed a method of producing HVJ from eggs and purifying it by the steps of filtration, membrane concentration, ultrafiltration, ion exchange chromatography, and ultrafiltration (TO03 / 014338). However, in this method, two treatments, filtration and membrane concentration, were put in front of the column, and this physical stimulation reduced the activity of HVJ, and it was inevitable that the recovery rate would be reduced due to trapping at this stage.

In W096 / 27677, an adenovirus purification method using an ion exchange resin and an immobilized metal affinity resin has been developed as a virus purification method. However, since adenovirus has no envelope, this method could not be used as a method for purifying enveloped virus. further As a method for purifying adenovirus, a method using an ion exchange resin and a hydrophobic resin in combination has also been proposed, but no specific examples have been disclosed. In addition, W091 / 00104 discloses a method for purifying the HN and F proteins of enveloped virus by affinity mouthmatography, but this is a virus envelope that is an aggregate of many proteins. It cannot be applied to the purification method.

 Therefore, in order to solve the above-mentioned problems and efficiently produce the virus envelope, it has been desired to develop a purification method having a higher recovery rate while maintaining the activity of HVJ. Disclosure of the invention

 An object of the present invention is to develop a method for purifying a virus envelope with a higher recovery rate and retaining the virus fusion activity.

 Envelope viruses, including HVJ, are complex proteins consisting of several proteins, lipids, and sugar chains, and are generally large particles with a diameter of 100 nanometers. Was expected to have high hydrophobicity. Therefore, designed for separating relatively small molecules, it has a smaller pore size (aperture per pore on the support surface), a smaller surface area per unit, and higher resolution. With the recent chromatography matrix support, various interactions between HVJ and the support surface could not be fully utilized. In other words, in order to separate a large and highly hydrophobic molecule such as HVJ, there is a gap between the loose pore size or sufficient surface area that HVJ can distribute inside the carrier and sufficient functional groups that HVJ can bind to. Interactions with distance, sufficient binding constant and appropriate dissociation constant were required.

The culture supernatant contains various impurities other than HVJ. However, most of them are molecules or particles smaller than HVJ. Most of today's chromatographic carriers have selectivity on the small molecule side. Therefore, they could not properly separate HVJ from the culture supernatant. Therefore, it was necessary to study a purification method suitable for HVJ. The present inventor has intensively studied to solve these problems. As a result, a purification method comprising a hydrophobic chromatography, preferably a combination of hydrophobic chromatography, anion exchange chromatography and / or gel filtration chromatography, has been established.

 That is, the present invention provides, as a means for solving the problems, a method for efficiently purifying at a high recovery rate while maintaining the cell fusion activity of the virus envelope.

The gist is

 (1) A method characterized by using hydrophobic chromatography for purification of the viral envelope,

 (2) The method according to (1), wherein the functional group of the hydrophobic matrix chromatography is a weakly hydrophobic group,

 (3) The method according to (1) or (2), wherein the functional group of the hydrophobic chromatography is an oligoethylene glycol group or a phenyl group,

(4) The method according to any one of (1) to (3), wherein the functional group of the hydrophobic chromatography is an oligoethylene glycol group,

 (5) The method according to any one of (1) to (4), characterized by combining the hydrophobic matrix chromatography and the ion exchange resin matrix and Z or gel filtration chromatography,

 (6) The method according to (5), characterized in that ion exchange chromatography and hydrophobic chromatography are combined.

 (7) The method according to (5) or (6), wherein an ion exchange resin is used for the first chromatography and a hydrophobic resin is used for the second chromatography.

(8) The method according to any one of (5) to (7), wherein the ion exchange chromatography is an anion exchange chromatography (9) The method according to any one of (5) to (8), wherein the ion exchange chromatography is a weak anion exchange chromatography.

 (10) The method according to any one of (5) to (9), wherein the functional group of the ion exchange chromatography is a jetylaminopropyl (DEAP) group,

 (11) The method according to any one of (5) to (10), wherein the functional group of the ion exchange chromatography is a low density substitution type (Low Sub),

 (1 2) The method according to (1) to (11), wherein the chromatography buffer has a pH of 7 to 9,

 (1 3) The method according to (1) to (: L2), wherein the chromatography buffer has a pH of 7.8 to 8.5,

 (14) The method according to any one of (5) to (13), wherein the buffer used for ion exchange chromatography includes sodium chloride or chloride power rhomium,

(15) The method according to any one of (5) to (14), wherein the buffer at the time of sample adsorption of ion-exchange chromatography includes sodium chloride.

 (16) The method according to any one of (5) to (15), wherein the buffer at the time of sample adsorption in ion exchange chromatography contains sodium chloride 30 to 70 mM.

 (17) The method according to any one of (5) to (16), wherein the buffer during washing of the ion exchange chromatography contains 150 mM to 250 mM sodium chloride,

 (18) The method according to any one of (5) to (17), wherein the buffer used for washing the ion exchange matrix chromatography contains 190 to 230 mM sodium chloride,

 (19) The method according to any one of (5) to (18), wherein the buffer at the time of elution of the ion exchange chromatography matrix contains sodium chloride lOOmM ~; LOOOmM,

(20) The method according to (5) to (19), wherein the buffer at the time of elution of ion exchange chromatography contains 290 to 330 mM sodium chloride,

(2 1) The method according to any one of (1) to (2 0), wherein the buffer used for hydrophobic chromatography contains ammonium sulfate, sodium sulfate or sodium chloride, (2 2) The method according to any one of (1) to (2 1), wherein the buffer at the time of adsorption of hydrophobic chromatography contains ammonium sulfate 1.0M to 2.5M,

 (2 3) The method according to any one of (1) to (2 2), wherein the buffer at the time of adsorption of hydrophobic chromatography contains ammonium sulfate 1, 8 to 2 · 2M,

 (2 4) The method according to (1) to (2 3), wherein the buffer at the time of washing by hydrophobic chromatography contains ammonium sulfate 1.0 to 1.6M,

 (2 5) The method according to (1) to (2 4), wherein the puffer at the time of washing the hydrophobic mouthmatography contains ham-sulfate 1.2 to 1.5 M,

 (2 6) The method according to (1) to (2 5), wherein the buffer at the time of elution of hydrophobic chromatography contains 0.01 to 1.5 M ammonium sulfate,

 (2 7) The method according to (1) to (2 6), wherein the buffer at the time of elution of hydrophobic chromatography contains ammonium sulfate 0.6 to 10 M,

 (2 8) The method according to any one of (1) to (2 7), wherein the buffer at the time of elution of hydrophobic chromatography contains a hydrophilic organic solvent,

 (2 9) The method according to (28), wherein the hydrophilic organic solvent is a polyhydric alcohol or a lower alcohol.

 (3 0) The method according to (2 8) or (2 9), wherein the polyhydric alcohol is ethylene glycol,

 (3 1) The method according to (2 8) to (3 0), wherein the concentration of ethylenedaricol is 0.01 to 50%,

 (3 2) The method according to (2 8) to (3 1), wherein the concentration of ethylene glycol is 2 to 10%,

 (3 3) The method according to (2 8) to (3 2), wherein the concentration of ethylene glycol is 3 to 7%,

 (3 4) The method according to any one of (1) to (3 3), wherein the buffer at the time of elution of hydrophobic chromatography ^-contains a surfactant,

( ₃₅ ) The surfactant is Tween 80 or _Triton . X (Triton X), the method according to (1) to (3 4),

 (36) The method according to (1) to (35), wherein the buffer at the time of elution of hydrophobic chromatography contains Tween 80 0.001-1%,

 (3 7) The method according to (1) to (3 6), wherein the buffer at the time of elution of hydrophobic chromatography contains Tween 80 0.01 to 0.1%,

 (3 8) The method according to any one of (1) to (3 7), characterized in that in hydrophobic chromatography, the sample is adsorbed and then eluted by lowering the temperature by 5 ° C or more from the adsorption temperature.

 (3 9) The method described in (3 8), wherein the temperature during elution is in the range of (room temperature 1-5) ° C to 4 ° C,

 (40) In the hydrophobic chromatography, after the sample is adsorbed, elution is carried out by raising the pH of the buffer at the time of adsorption by 0.5 or more, (1) to the method according to (39),

 (4 1) The method according to (4 0), wherein the pH at the time of elution is in the range of 6 to 10; (4 2) Gel filtration chromatography is performed following hydrophobic chromatography, (1) to (4 1) described method,

 (4 3) The method according to any one of (1) to (4 2), wherein the carrier for gel filtration chromatography is sepharose,

 (4 4) A method according to (1) to (4 3), wherein a divalent metal ion is added to a buffer used for gel filtration chromatography.

 (4 5) The divalent metal ion is calcium or magnesium, (4

4) described method,

 (4 6) The divalent metal ion concentration is 0.1 to 10 mM, (4 4) or (4

5) described method,

 (4 7) The divalent metal ion concentration is; ~ 2mM, (4 4) ~ (4 6) described method,

(4 8) Winoles is Buirouinores, Punyawinoles, Henorepesu Inoresu Department, box Huy / Les Department, togaviruses family, corona virus family, the Flaviviridae family, paramyxovirus family, Arenawirusu family, ortho Mikusoui / Les Department, c ⁰ Donauinoresu Department of Les DOO Roui / Les Department, to, Les old Huy The method according to (1) to (47), which is a virus belonging to a family selected from the group of Rusaceae and Deltaviridae,

 (4 9) The virus is Ebola virus, Crimea Congo hemorrhagic fever wi-nores, Nounta wi-nores, Herpes simplex enoures, EB quinoles, Natural pox virus, Cowpox virus, Rubella virus, SARS virus (Hitocorona virus), C Hepatitis B virus, Japanese encephalitis virus, yellow fever virus, dengue virus, West Nile virus, Russian spring and summer encephalitis virus, swine fever virus, rabies virus, vesicular stomatitis virus, Sendai virus (HVJ), measles virus, Mumps virus, mumps virus, rubella virus, RS virus, Lassa fever virus, influenza virus, human immunodeficiency virus (HIV), human T cell leukemia type I virus (HTLV-1), feline immunodeficiency virus ( FIV), hepatitis B virus (HBV), Virus is a virus selected from the group of hepatitis D virus (1) to (4 8 ') The method according,

 (50) The method according to (1) to (49), wherein the virus is HVJ,

 (5 1) The method according to any one of (1) to (50), wherein the virus envelope is an attenuated or inactivated virus envelope,

 (5 2) The method according to any one of (1) to (5 1), wherein the virus envelope is an inactivated winores envelope,

About.

Moreover, the virus envelope purified by the present invention can be widely used as a vector for introducing a low-molecular or high-molecular compound into cells or living organisms. For example, as disclosed in W02004 / 039406, a chemotherapeutic agent can be encapsulated and used as an anticancer agent. Also As shown in W02004 / 046353, any nucleic acid can be encapsulated and used for screening the target gene or protein. Brief Description of Drawings

FIG. 1 is a diagram showing an anion exchange chromatogram of column 1 (Example 1).

FIG. 2 shows a hydrophobic chromatogram of column 2 (Example 1). FIG. 3 is a diagram showing a gel filtration matrix of column 3 (Example 1). FIG. 4 is a diagram showing an electrophoretic image of a purified HVJ envelope SDS polyacrylamide (Example 3). Lane 1: Molecular weight marker, Lane 2: Cell-derived HVJ (reduced), Lane 3: Chicken egg-derived HVJ (reduced), Lane 4: Cell-derived HVJ (non-reduced), Lane 5 _: Chicken egg-derived HVJ (non-reduced). BEST MODE FOR CARRYING OUT THE INVENTION

 As used herein, “gene transfer” means in vivo or in vitro the desired gene or gene fragment, natural, synthetic or recombinant, and its function in the target cell. Say to introduce to maintain. The gene or gene fragment introduced in the present invention includes a nucleic acid that is DNA, RNA, or a synthetic analog thereof having a specific sequence. In addition, as used herein, gene transfer, transformation, and transformate are used interchangeably.

As used herein, “gene vector”, “gene transfer vector”, or “winores envelope vector” refers to a vector in which a foreign gene is encapsulated in a winores envelope. The virus used for the preparation of the gene transfer vector may be a wild type virus or a recombinant virus. In one aspect of the present invention, the viruses used are: Firoviridae, Bunyawinoles, Henorepesuinores, Boxwirs, Togaviridae, Coronaviridae, Flaviviridae, Noramixuinolesidae, A virus belonging to a family selected from the group consisting of the genus Arenawinoles, the genus Noreto Myxoinoles, the Letrouinoleceae, the Hepadnaviridae, the Reoviridae, and the Deltaviridae.

 Preferably, these viruses are Ebola virus, Crime-Congo hemorrhagic fever wi-nores, non-tuino hinoles, simple herpes peinos, EB virus, smallpox virus, cowpox virus, rubella virus, SARS virus (Hitocoronavirus) ), Hepatitis C virus, Japanese encephalitis virus, yellow fever virus, dengue virus, West Nile virus, Russian spring / summer encephalitis virus, swine fever virus, rabies virus, vesicular stomatitis virus, Sendai virus (HVJ), measles virus Mumps virus, mumps wingless, rubella virus, rs wingless, lassa funo wingless, influenza virus, human immunodeficiency virus (HIV), human T cell leukemia type I virus (HTLV-1), feline immunity Incomplete virus (FIV), B Hepatitis Virus (HBV), a reovirus, a virus that is selected from the group consisting of hepatitis D virus.

 More preferably, the virus is HVJ.

 As used herein, “inactivated virus” refers to a virus that has inactivated its genome. This inactivated virus is replication defective. Preferably, this inactivation is done by UV treatment or treatment with an alkylating agent.

 As used herein, an “attenuated virus” refers to a virus that infects a host with little or no pathogenicity.

As used herein, “foreign gene” refers to a nucleic acid sequence derived from other than a virus contained in a gene transfer vector. One station of the present invention In terms of this, this foreign gene can be a regulatory gene suitable for the expression of the gene introduced by the gene transfer vector (eg, a promoter, enhancer, terminator, and poly A addition signal required for transcription, and translation). The ribosome binding site, the start codon, the stop codon, etc.) In another aspect of the present invention, the foreign gene does not contain a regulatory sequence for expression of the foreign gene. In a further aspect of the invention, the foreign gene is an oligonucleotide or a deconucleic acid. The foreign gene contained in the gene transfer vector is typically a DNA or RNA nucleic acid molecule, but the nucleic acid molecule to be introduced may contain a nucleic acid analog molecule. The molecular species contained in the gene transfer vector may be a single gene molecular species or a plurality of different gene molecular species.

 In the present specification, “HVJ” and “Sendai virus” are used interchangeably.

 As used herein, “HAU” refers to the activity of a virus capable of aggregating 0.5% of chicken erythrocytes, and 1 HAU corresponds to approximately 24 million virus particles (Okada, Y et al., Biken Journal 4, 209-213, 1961).

 The purification method of the present invention is a force that can be achieved only by hydrophobic chromatography, preferably hydrophobic chromatography, ion exchange chromatography and / or gel filtration chromatography, specifically the following combinations: Includes one of the combinations.

 (1) Hydrophobic chromatography

 (2) Combination of hydrophobic mouthmatography and ion exchange mouthmatography (in no particular order)

 (3) Combination of hydrophobic chromatography and gel filtration chromatography (in no particular order)

(4) Hydrophobic chromatography and ion exchange chromatography Combination of filtration chromatography (in no particular order)

Most preferably, a three-step column chromatography is used. However, the third step is intended for desalination and concentration, so it is performed as necessary. PH of the buffer used for the chromatography one can use the usual _{P H7~9.} Preferably, pH 7.8 to 8 · 5 is used.

Column 1 can usually be an anion exchange chromatography. Theoretically, almost all of the anion exchanger (DEAP, Q, QAE, DEAE, etc.) Allowed for force ^¾ flights affirmative Demel (DEAP; di ethyl aminopropyK Q; quaternary amine ^ QAE; quaternary aminoethyl DEAE; di ethylaminoethyl). As an example of Tana, for example, ANX- Sepharose 4FF (GE Amersham Biosciences, Cat. No. 17-1286-01) with DEAP can be cited. This exchanger is a carrier specialized for capturing macromolecules by the relatively large pore size and the distance between the carrier and the functional group by the propyl group of the DEAP group becoming a spacer. ANX- Sepharose4FF Low Sub also increases selectivity from small molecules to large molecules by reducing the functional group density. This is because chromatographic interactions usually involve multiple functional groups attached to a protein molecule. Common chromatographic supports bind a large excess of functional groups, resulting in a very dense environment. Since the carrier has a structure in which the strings are entangled, it becomes an advantageous environment for a low molecule that can be bound by more points in the space. In other words, ANX- Sepharose 4FF improved the three-dimensional space of functional groups for polymers. As the buffer used for the ion exchange chromatography, a buffer having a pH of 6 to 9 can be used, and examples thereof include Tris-HCl buffer, phosphate buffer, and HEPES buffer.

Column 2 can then usually be used with hydrophobic chromatography. As an example of a carrier used for hydrophobic chromatography, among the commercially available hydrophobic carriers, those having weak hydrophobicity are preferable. For example, Ether And a hydrophobic carrier having an Alkyl group, and a carrier having an Ether group is particularly preferred. As a preferred specific example, Ether-TOYOPEARL650M (Cat. No. 16173) manufactured by Tosoh has an oligoethylene glycol group as its functional group. Oligoethylenedaricol group is considered to have weak hydrophobicity because it has 0H side chain in the carbon chain. This is because proteins with high hydrophobicity are not sufficiently separated from each other with the commonly used hydrophobic chromatography media Butyl group and Pheny group, because the binding force between them is too strong, resulting in lower recovery. It was developed for this purpose. In addition, the functional group of Toyopearl is bound by a method that does not involve a spacer, so it appears to be even less hydrophobic than the products of other companies. This can also be predicted from data when comparing Phenyl- Sepharose (with a spacer) and Phenyl-TOYOPEARL. Normally, it is difficult to purify viruses using a hydrophobic chromatography with a Butyl group, Phenyl group, etc., but smaller functional viruses can be used when functional groups are made less dense than normal products (commercially available products). But it can be purified. A buffer of pH 6-9 can be used as a buffer for hydrophobic chromatography. For example, Tris-HCl buffer, phosphate buffer, HEPES buffer, B is-Tris / HC 1, B is — Trispropane / HC 1, Trietanolamine / HC 1, Triethanolamine Z Acetic Acid, N-Methyljetanolamine / HC 1, N-Methyljetanolamine Z Acetic Acid, Diethanolamine / HC 1, 1,3-Diaminopropane / HC1, piperazine ZHC1, trimethylamine ZHC1, ethanolamine / HC1, n-methylmorpholine ZHC1, etc. HVJ bound to oligoethylene glycol groups elutes by reducing the ammonium sulfate concentration in the buffer, but that alone does not sharpen the peaks and requires a considerable amount of liquid to completely elute. . Therefore, it is effective to add a hydrophilic organic solvent to increase the polarity of the solution. The hydrophilic organic solvent is It can be added in the range of 0.01 to 50%. This concentration is not limited, but is usually 2% to 10%, preferably 3 to 7%, and most preferably 5%. As the hydrophilic organic solvent, a polyhydric alcohol or a lower alcohol is preferably used. The lower alcohol in the present invention is not limited as long as it is an alcohol in which one hydroxyl group is added to a lower hydrocarbon having 1 to 6 carbon atoms. Specifically, for example, methanol, ethanol, n-propanol, i-prono Nonorre, n-ptanol monole, i-ptanol monole, t-peptanol etc. The polyhydric alcohol is not limited as long as it is an alcohol in which two or more hydroxyl groups are added to a hydrocarbon having 2 to 6 carbon atoms. Specifically, for example, ethylene glycol, propylene dalycol, trimethylene glycol, Examples thereof include glycerin. More preferably, a polyhydric alcohol is used. Most preferably, ethylene glycol is used. Ethylene glycol can be added in the range of 0.01 to 50%. This concentration is not limited, but is usually 2% to 10%, preferably 3 to 7%, and most preferably 5%. When ethylene glycol is added, the peak becomes sharp and the elution volume is about half of the force ram volume, so that purification and concentration can be performed simultaneously. In this step, HVJ may bind to each other because it is purified at a high ammonium sulfate concentration. In order to prevent this binding, it is effective to add a surfactant to the elution buffer. The surfactant can be added in the range of 0.001 to 1%, preferably in the range of 0.01 to 0.1%. As the surfactant, Tween or Triton X can be used. More preferably, Tween 80 is used. Tween 80 can be added in the range of 0.001 to 1 ° / ₀ . Tween 80 is preferably added in an amount of 0.01 to 0.1%. Most preferably, 0.05% is added.

Further, gel filtration chromatography can be usually used for column 3. This step is used for concentration and desalination, so it is widely used Gel filtration chromatography can be used. For example, Sepharose 4 FF (Cat. No. 17-0149-01) and Sepharose 6 FF (Cat. No. 17-0159-01) from GE Amersham Bai Science can be used. As a buffer for gel filtration, pH 6-9 buffer can be used. For example, Tris-HCl buffer, phosphate buffer, HEPES buffer, Bis — Tris / HC 1, Bis-Tris propane / HC 1, triethanolamine / HC 1, triethanolamine / acetic acid, N-methyl diethanolamine ZHC 1, N-methyljetanolamine / acetic acid, diethanolamine / HC 1, 1, 3— Examples include diaminopropane / HC 1, piperazine / HC 1, trimethylamine ZHC 1, ethanolamine / HC 1, n-methylmorpholine / HC 1, and the like. It is to be noted that a hydrophilic organic solvent may be added to the buffer, and examples of the hydrophilic organic solvent that can be added include those described above.

 It is also effective to add a divalent metal ion as a stabilizer for neurominidase to the buffer and / or final purified product used in gel filtration chromatography. The divalent metal ion is preferably calcium or magnesium. Usually, these metal ions are added in the range of 0.1 to 1 OmM. Preferably, it is added to a concentration of 1 to 2 mM. Example

 Subsequently, in order to specifically describe the present invention, examples are given below, but the present invention is not limited thereto. Example 1. Purification of HV.T by column chromatography

The HVJ produced by the suspension cell culture system was purified from the cell culture supernatant obtained by removing the cell by centrifugation and purified by a three-stage chromatography process. First, in order to inactivate endotoxins, each column was used after passing through a 0.25 M sodium hydroxide solution and holding for 8 hours or more before use.

 (1) Ion exchange resin chromatography

 The centrifuged culture supernatant was diluted 1: 5 with 5 OmM Tris-HCl buffer pH 7.8. This was passed through column 1 equilibrated with 50 mM Tris-HCl buffer pH 7.8 containing 50 mM sodium chloride and adsorbed. Column 1 contains an anion exchange chromatography carrier, AN X—Sepharose 4 FFLow Sub (GE Amersham Biosciences, Cat. No. 1 7-1 2 8 6-0 1) BPG 2 00/5 00 empty column (GE Amersham Biosciences) 3 L packed 20 cm in diameter and 9 centimeters high was used. The linear flow rate was 96 cm / h. After passing through the equilibration buffer of 3 times the column volume, 50 mM Tris-HCl buffer solution pH 7.8 containing 8 times the column volume of 2 1OmM sodium chloride was passed through. And washed. To elute HVJ from column 1, 50 mM Tris-HCl buffer (pH 7.8) containing 310 mM sodium chloride was passed. At this time, the absorbance at UV 280 nm of the column passing solution increased, so that portion was recovered. This was used as the column 1 elution fraction. Finally, column 1 was regenerated by passing 50 mM Tris-HCl buffer containing 2 M sodium chloride. (refer graph1)

 (2) Hydrophobic resin chromatography

Next, an equal volume of 4 M ammonium sulfate solution was added to the column 1 elution fraction to prepare a solution containing 2 M ammonium sulfate. The solution was passed through column 2 equilibrated with 50 mM Tris-HCl buffer solution pH 7.8 containing 2M ammonium sulfate and adsorbed. Column 2 contains E ther -TOYO P EAR L 6 50 M (Tosohichi Co., Ltd., Cat. No. 1 6 1 7 3), a carrier for hydrophobic interaction chromatography, BPG 1 00/5 00 Empty column (GE 1 sq. Filled with 10 cm diameter and 12.5 cm height was used. The linear flow rate was 190.000 mZh. Further, the equilibration buffer of 3 volumes of the column volume was passed through, and then washed by passing 50 mM of Tris-HCl buffer containing 1.4 M ammonium sulfate of 5 volumes of the column volume. For elution of HVJ from column 2, 50 mM Tris-HCl buffer pH 7.8 containing 0.8 M ammonium sulfate, 5% ethylene glycol, and 0.05% Tween 80 was passed. . At this time, the absorbance at UV 2800 nm of the force ram passing solution increased, and that portion was recovered. This was used as the column 2 elution fraction. Finally, 5% ethylene glycol solution was passed through to regenerate column 2. (See Figure 2)

 (3) Gel filtration chromatography

 Further, the fraction eluted from column 2 was passed through column 3 equilibrated with 50 mM Tris-HCl buffer solution pH 7.8 containing 150 mM sodium chloride. Column 3 contains Sepharose 4 FF (manufactured by GE Amersham Biosciences, Cat. No. 1 7-0 1 4 9— 0 1), a carrier for gel filtration chromatography, XK 5 0Z6 0 empty column ( GE Amersham Biosciences) with a diameter of 5 cm and a height of 40 cm was used. The linear flow rate was 120 cmzh. Next, the equilibration buffer was passed through the column elution fraction. At this time, the absorbance at UV 2880 nm of the force ram passing solution increased, and the portion was recovered. This was the column 3 elution fraction. (See Figure 3)

 An HVJ solution containing 0.1% methylcellulose in a final concentration of 0.1% was prepared by adding 0.5% methylcellulose in an amount equivalent to 1 volume of 4% to the column 3 elution fraction. This was filtered through a 0.45 micron sterile filtration filter. By rapidly freezing this in liquid nitrogen and storing it at 180 ° C, high-purity HVJ could be stably stored for a long time.

Main reagent used for buffer etc. Tris (Hydroxymethyl) Minomethane, sodium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydroxide, ethylene glycol, ammonium sulfate, and Tween 80 are manufactured by Wako Pure Chemicals or Sigma Special grades made by the company were used. Hydrochloric acid was a 6 standard product manufactured by Wako Pure Chemical Industries. The 0.5% methylcellulose added as a stabilizer for purified products was manufactured by Wako Pure Chemical Industries. For aseptic filtration of the purified product, a hydrophilic filter filter Millipak Gamma a Gold (Nippon Millipore) with a pore size of 0.45 microns was used. Comparative Example 1. Purification of HV. Ding by membrane concentration and column chromatography Cell culture supernatant obtained by cell removal treatment by centrifugation of HVJ produced by floating cell culture system was concentrated to the outer membrane. And three-stage kumatography.

 First, the membrane concentrator and each column were used after passing through a 0.25 M sodium hydroxide solution and holding for 8 hours or more before use in order to inactivate endotoxin (typically Holding for about a week can completely remove viable bacteria and endotoxin).

 (1) Filtration and membrane concentration

 The centrifuged culture supernatant was filtered through a 1.2-m milliguard filter to remove particles. This was concentrated to 600 mL using a pelicon membrane concentrator.

 (2) Gel filtration chromatography (Column 1)

The concentrated culture supernatant obtained in (1) was passed through column 1 equilibrated with 50 mM Tris-HCl buffer pH 7.8 containing 150 mM sodium chloride. Column 1 contains Sephar ο se 6 FF (GE Amersham Biosciences), a carrier for gel filtration chromatography, and BPG 1 0 0/5 0 0 empty column (GE Amersham Biosciences) diameter 10 SE Inch, 2.5 L filled with a height of 33 cm was used. The linear flow rate was 1550 cmZh. The equilibrated puffer was then passed through. At this time, the absorbance at UV 2880 nm of the column passing solution increased, and a portion thereof was recovered. This was used as the column 1 elution fraction.

 (3) Ion exchange chromatography (Column 2)

 Next, the column 1 elution fraction 1 was passed through column 2 equilibrated with 5 O mM Tris-HCl buffer pH 7.8 containing 50 mM sodium chloride and adsorbed. Column 2 is Q—Sepharose FF (GE Amersham Biosciences), which is a carrier for ion exchange chromatography, and BPG 200/500 aerodynamic ram (GE Amersham Biosciences), 20 cm in diameter, high A 2 L container filled to 6.5 cm was used. The linear flow rate was 70 cm / h. Furthermore, one column of equilibration buffer of 5 times the column volume was passed. For elution of HVJ from column 2, 50 mM Tris-HCl buffer solution pH 7.8 containing 0.45 M sodium chloride was passed. At this time, the absorbance at UV 28 80 nm of the force ram passing liquid increased, and the portion was recovered. This was used as the column 2 elution fraction. Finally, 2M sodium chloride was passed through to regenerate column 2.

 (4) Gel filtration chromatography (Column 3)

The fraction eluted from column 2 was passed through column 3 equilibrated with 50 mM Tris-HCl buffer pH 7.8 containing 150 mM sodium chloride. Column 3 contains Sepharose 6 FF (manufactured by GE Amersham Biosciences), a gel filtration chromatographic carrier, and BPG 1 00Z 5 00 aerodynamic ram (manufactured by GE Amersham Biosciences), diameter 10 cm, height 3 A 2.5 L container filled with 3 cm was used. The linear flow rate was 1550 cm Zh. The equilibration buffer was passed through the column elution fraction. At this time, the absorbance at UV 2800 nm of the column passing solution increased, and the portion was recovered. This was the column 3 elution fraction. A 1/4 volume equivalent of 0.5% methylcellulose was added to the column 3 eluate to prepare a HVJ solution containing a final concentration of 0.1% methylcellulose. This was filtered through a 0.45 micron sterile filtration filter.

Main reagents used for buffer etc. Tris (Hydroxymethyl) aminomethane, Sodium chloride, Calcium chloride dihydrate, Magnesium chloride hexahydrate, Sodium hydroxide are made by Wako Pure Chemical, or A special product manufactured by Sigma was used. Hydrochloric acid was a 6 standard product manufactured by Wako Pure Chemical Industries. The 0.5 ° / ₀ methylcellulose added as a stabilizer for the purified product was manufactured by Wako Pure Chemical Industries. For aseptic filtration of purified products, a Milliak G amm a Gold (Japan Millipore) with a pore size of 0.45 microns was used. Example 2. Activity measurement and recovery of purified HV.T

The activity of HVJ was measured by sialic acid degrading enzyme (neuraminidase, NA) activity and agglutination activity of chicken erythrocytes (hemadalchunic activity, HA). Both are proportional to the concentration and amount of recovered virus, and are also quantitative. Table 1 shows the residual activity of HVJ in Example 1 of the present invention by these two methods and the recovery rate by NA activity value. Table 2 shows the recovery rate when the comparative example was used. Comparison of Tables 1 and 2 clearly shows the excellent effect of the present invention.

(Table 1) (Invention, Example 1) Step Volume (mL) NA activity (mU / tnL) Total NA (mU) Recovery (NA%) HA (U) Starting material 10250 297. 0 3044066 100. 0

Column 1 1200 1891. 4 2269735 74. 6

Column 2 450 3524. 0 1585790 52. 1

Column 3 500 3627. 6 1813813 59. 6

Filtration 625 3027. 2 1892022 62. 2

End product

 (Inactivated virus) 590 2066. 1 1218993 40. 0 5120

(Table 2) (Conventional purification method, Comparative example 1) Step volume (mL) NA activity (mU / mL) Total NA (mU) Recovery (NA%) HA (U) Starting material 10800 71. 9 775982 100. 0

Filtration 10800 51. 1 551397 71. 1

Membrane concentration 600 903. 4 542069 69. 9

Column 1 1000 573. 4 573351 73. 9

Column 2 1000 373. 8 373770 48. 2

Column 3 1050 393. 8 413483 53. 3

Filtration 1300 218. 3 283804 36. 6

End product

(Inactivated virus) 1 120 159. 7 178864 23. 1 2560 Example 3. Purification by SDS-PAGE Purity analysis of HVJ

 HVJ electrophoresis was performed on SDS-PAGE gel with 4 1 1 2 Q / o. HVJ was solubilized using a membrane protein solubilizer suitable for viruses (Sigma CelLyt ic— M Cat. No. C2978) and electrophoresed as it was. A reduction sample was obtained by adding mercaptoethanol and heating at 95 ° C for 10 minutes. As a comparison with the general method, HVJ was purified by anion exchange chromatography after culturing in fertilized chicken eggs and treated in the same way. This was electrophoresed at a constant voltage of 100 V for 2.5 hours, stained with SYPROR uby fluorescent staining solution (SYPRO Ruby Protein Gel Stain Cat. No. 170-3125), and then using a fluorescence imaging system. The gel was analyzed. The results of electrophoresis are shown in FIG. Compared with the conventional purification method from chicken eggs, the HVJ envelope with the same purity could be obtained. Industrial applicability

 It is possible to provide a method for purifying a viral envelope with high recovery rate while maintaining its cell fusion activity. The purified viral envelope can be used as a vector for introducing a biopolymer such as a gene into a cell or a living body.

By using the method of the present invention, an inactive virus envelope (for example, HVJ) can be purified while retaining its fusion activity at a higher recovery rate than in the conventional method. Therefore, it is useful for industrial production of virus envelopes. The method of the present invention can also be applied to the purification of non-inactivated envelope virus. This application is based on Japanese Patent Application No. 2 0 0 4— 2 1 9 3 8 1 filed in Japan (filing date: July 2, 7th, 2000), the contents of which are incorporated herein by reference. It is included.

Claims

The scope of the claims  I. A method characterized by using hydrophobic chromatography in the purification of the viral envelope.  2. The method according to claim 1, wherein the functional group of the hydrophobic chromatography is a weakly hydrophobic group.  3. The method according to claim 1 or 2, wherein the functional group of the hydrophobic matrix is an oligoethylene glycol group or a phenyl group.  4. The method according to any one of claims 1 to 3, wherein the functional group of the hydrophobic matrix is an oligoethylene glycol group. 5. The method according to any one of claims 1 to 4, wherein the hydrophobic chromatography and ion exchange resin chromatography are combined with Z or gel filtration chromatography.  6. The method according to claim 5, characterized in that ion exchange chromatography and hydrophobic chromatography are combined. 7. The method according to claim 5 or 6, wherein an ion exchange resin is used for the first chromatography and a hydrophobic resin is used for the second chromatography.  8. The method according to claims 5 to 7, wherein the ion exchange matrix is an anion exchange matrix.  9. The method according to claims 5 to 8, wherein the ion exchange chromatography is weak anion exchange chromatography.  10. The method according to any one of claims 5 to 9, wherein the functional group of the ion exchange chromatography is a jetylaminopropyl (DEAP) group.  I I. The method according to claims 5 to 10, wherein the functional group of the ion exchange chromatography is of a low density substitution type (Low Sub). 12. The method according to any one of claims 1 to 11, wherein the chromatographic buffer has a pH of 7-9. 1 3. Claim that the chromatography buffer has a pH of 7.8 to 8.5. The method according to any one of Items 1 to 12.  14. The method according to any one of claims 5 to 13, wherein the buffer used for ion exchange chromatography contains sodium chloride or power chloride. 15. The method according to any one of claims 5 to 14, wherein the buffer at the time of adsorption of the sample of the ion exchange chromatography matrix contains 0.01 to 150 mM sodium chloride. 16. The method according to any one of claims 5 to 15, wherein the buffer for adsorbing the sample of ion exchange chromatography contains 30 to 70 mM sodium chloride. 17. The method according to any one of claims 5 to 16, wherein the buffer during washing of the ion exchange chromatography contains 150 mM to 250 mM sodium chloride. 18. The method according to any one of claims 5 to 17, wherein the washing buffer for ion exchange chromatography contains 190 to 230 mM sodium chloride. 19. The method according to any one of claims 5 to 18, wherein the buffer at the time of elution of the ion exchange chromatography contains from 100 mM to 1000 mM sodium chloride. 20. The method according to any one of claims 5 to 19, wherein the puffer at the time of elution of the ion exchange chromatography contains 290 to 330 mM sodium chloride.  21. The method according to any one of claims 1 to 20, wherein the buffer used in the hydrophobic chromatography comprises ammonium sulfate, sodium sulfate or sodium chloride. .  2. The method according to claim 1, wherein the buffer for adsorption of the hydrophobic chromatography contains ammonium sulfate 1.0 M to 2.5 M.  2 3. The method according to claim 1 or 2, wherein the buffer for adsorption of the hydrophobic chromatography contains ammonium sulfate 1.8 to 2.2M. 24. The method according to any one of claims 1 to 23, wherein the buffer during washing of the hydrophobic chromatography comprises ammonium sulfate 1.0 to 1.6M. 25. The method according to any one of claims 1 to 24, wherein the buffer for washing of the hydrophobic chromatography contains ammonium sulfate 1.2 to 1.5M. 2 6. The buffer used for the elution of hydrophobic chromatography is ammonium sulfate. Method according to claims 1 to 25, comprising rum 0.01 to 1.5M.  27. The method according to any one of claims 1 to 26, wherein the buffer used for the elution of the hydrophobic chromatography comprises ammonium sulfate 0.6 to 1.0M.  28. The method according to any one of claims 1 to 27, wherein the buffer at the time of elution of the hydrophobic matrix chromatography contains a hydrophilic organic solvent.  29. The method according to claim 28, wherein the hydrophilic organic solvent is a polyhydric alcohol or a lower alcohol.  30. A process according to claim 28 or 29, wherein the polyhydric alcohol is ethylene glycol. 3. The method according to claim 28, wherein the concentration of ethylene glycol is 0.01 to 50%.  3. The method according to claim 28, wherein the concentration of ethylenedaricol is 2 to 10%.  3 3. The method according to claim 28, wherein the concentration of ethylenedaricol is 3 to 7%.  3. The method according to any one of claims 1 to 33, wherein the buffer at the time of elution of the hydrophobic chromatography contains a surfactant.  35. The method of claims 1 to 34, wherein the surfactant is Tween 80 or Triton X. 36. The method of claims 1 to 35, wherein the buffer during elution of the hydrophobic '! "Raw chromatography comprises Tween 80 0.001 to 1 ° /. 37. The method according to any one of claims 1 to 36, wherein the buffer at the time of elution of the hydrophobic chromatography comprises Tween 80 0.01 to 0.1%. 3 8. The method according to claims 1 to 37, wherein in hydrophobic chromatography, the sample is adsorbed and then eluted by lowering the temperature by 5 ° C or more from the temperature at the time of adsorption. 3 9. The elution temperature is in the range of (room temperature 1 5) ° C to 4 ° C. 3 Method according to 8.  40. The method according to any one of claims 1 to 39, wherein in hydrophobic chromatography, after the sample is adsorbed, elution is performed by raising the pH of the buffer during adsorption by 0.5 or more.  41. The method according to claim 40, wherein the pH at elution is in the range of 6 to 10. 4. The method according to any one of claims 1 to 41, wherein the gel filtration chromatography is performed following the hydrophobic chromatography.  4 3. The method according to claims 1 to 42, wherein the carrier for gel filtration chromatography is sepharose. 4 4. The method according to claims 1 to 43, wherein a divalent metal ion is added to a buffer used for gel filtration chromatography. 4. The method according to claim 4, wherein the divalent metal ion is calcium or magnesium.  46. The method according to claim 44, wherein the concentration of divalent metal ions is 0.1 to 10 mM.  47. The method according to any one of claims 44 to 46, wherein the divalent metal ion concentration is 1 to 2 mM.  4 8. Winores is a philouinores, bujainoles, henorepesuinoles, boxwinores, togawinoles, coronawinoles, flaviviridae, noramixoviridae, arenaviridae, orthomixoviridae, retro The method according to any one of claims 1 to 47, which is a virus belonging to a family selected from the group of Viridae, Hepadnaviridae, Leovirus, and Deltaviridae. 4 9. Virus is Ebola virus, Crimea Congo hemorrhagic fever virus, Hunter virus, Herpes simplex puffin, EB quinoles, smallpox quinoles, cattle quinoles, rubella vinores, SARS vinores Hepatitis B virus, Japanese encephalitis virus, yellow fever virus, dengue Fever virus, West Nile virus, Russian spring and summer encephalitis virus, Putacholera virus, Rabies virus, Bullous stomatitis virus, Sendai virus (HVJ), measles virus, mumps virus, mumps virus, rubella virus, RS virus, Lassa virus, influenza virus, human immunodeficiency virus (HIV), human T cell leukemia type I virus The method according to any one of claims 1 to 48, wherein the virus is selected from the group consisting of (HTLV-1), feline immunodeficiency virus (FIV), hepatitis B virus (HBV), reovirus, and hepatitis D virus.  50. The method according to claims 1 to 49, wherein the virus is HVJ.  5. The method according to any one of claims 1 to 50, wherein the virus envelope is an attenuated or inactivated virus envelope.  5. The method according to claims 1 to 51, wherein the virus envelope is an inactivated virus envelope.